Method for improved production of cyanophycin and secondary products thereof

ABSTRACT

The present invention relates to a thermostable cyanophycin synthetase produced from  Synechococcus elongatus  and to a method for improved production of cyanophycin and/or secondary products thereof.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to a thermostable cyanophycinsynthetase, to transformed organisms containing such an enzyme and to amethod for improved production of cyanophycin and/or secondary productsthereof, for example polyaspartic acid or arginine.

BACKGROUND OF THE INVENTION

[0002] Multi-L-arginyl-poly-L-aspartate (cyanophycin) is a branchedpolypeptide which contains aspartic acid and arginine in the ratio of1:1. The chemical structure corresponds to a poly-α-aspartate backbonewith arginine side radicals which are linked via peptide bonds tovirtually all β-carboxyl groups of the backbone. DE-A 198 25 509describes the identification, cloning and heterologous expression of thegene for cyanophycin synthetase from Synechocystis PCC 6803. The enzymeactivity is determined here by means of a radioactive assay in whichL-[U-¹⁴C]-arginine is incorporated into cyanophycin from Aphanocapsa PCC6308, introduced as primer. The enzyme reaction itself takes place at28° C. here.

[0003] DE-A 197 09 024 discloses the extraction and purification ofcyanophycin from Aphanocapsa PCC 6308, the synthesis being carried outat 20° C.

[0004] DE-A 198 13 692 merely discloses isolation of the cyanophycinsynthetase gene from Synechocystis PCC 6803 or Anabaena variabilis ATCC29 413. Technical aspects of cyanophycin production, however, are notdescribed here.

[0005] FEMS Microbiology Letters 181 (1999) 229-236 discloses theproduction of cyanophycin using Synechococcus sp. MA 19.

[0006] A disadvantage of large-scale cyanophycin production according tothe known methods is that, for optimal product yield, a relativelynarrow temperature range, normally below 35° C., should not be exceeded.

[0007] This represents a considerable restriction in the degrees offreedom for large-scale production within the process control for theproduction of cyanophycin, since higher temperatures from the outsetprevent contamination by foreign cultures.

[0008] Therefore production of cyanophycin also at substantially highertemperatures than previously described, in combination with higherflexibility in process control and considerably improved product yieldsis desirable, in order to isolate therefrom the secondary products suchas polyaspartic acid or arginine on a large scale.

[0009] This object is achieved by the present invention.

SUMMARY OF THE INVENTION

[0010] The present invention relates to a cyanophycin synthetase whichis distinguished by having a temperature optimum in the range of >35° C.and an amino acid sequence according to SEQ ID No: 01, encoded by anisolated nucleotide sequence according to SEQ ID No: 02, an allele,homologue or derivative of this nucleotide sequence or a nucleotidesequence hybridizing therewith.

[0011] In a preferred variant of the present invention, the cyanophycinsynthetase of the invention has a temperature optimum in the range from35° C. bis 55° C., preferably in the range from 35° C. bis 50° C.

[0012] The cyanophycin synthetase is further distinguished by the factthat it originates from Synechococcus elongatus. The cyanophycinsynthetase of the invention represents a thermostable enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a representation of the chemical structure of thesynthetic peptide primers used for synthesis of cyanophycin by means ofcyanophycin synthetase.

[0014]FIG. 2 is a representation of the results of an SDS polyacrylamidegel electrophoresis (SDS-PAGE) for in vitro synthesis ofcyanophycin-like material by means of purified cyanophycin synthetase.

[0015]FIG. 3 is a representation of the results of an SDS-PAGE for chainelongation of a primer by means of cyanophycin synthetase at theC-terminal end of the peptide primer.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0016] The present invention relates to a cyanophycin synthetase whichis distinguished by having a temperature optimum in the range of >35° C.and an amino acid sequence according to SEQ ID No: 01, encoded by anisolated nucleotide sequence according to SEQ ID No: 02, an allele,homologue or derivative of this nucleotide sequence or a nucleotidesequence hybridizing therewith.

[0017] In a preferred variant of the present invention, the cyanophycinsynthetase of the invention has a temperature optimum in the range from35° C. bis 55° C., preferably in the range from 35° C. bis 50° C.

[0018] The cyanophycin synthetase is further distinguished by the factthat it originates from Synechococcus elongatus. The cyanophycinsynthetase of the invention represents a thermostable enzyme.

[0019] The present invention also relates to isoenzymes of thecyanophycin synthetase of the invention. These isoenzymes mean enzymeshaving identical or comparable substrate specificity and actionspecificity, but having a different primary structure. In addition, thepresent invention also includes modified forms of cyanophycinsynthetase. According to the invention, these mean enzymes in whichalterations are present in the sequence, for example at the N and/or Ctermini of the polypeptide or in the region of conserved amino acids,which alterations, however, do not impair the function of the enzymes.These modifications may be carried out by exchanging one or more aminoacids according to known methods.

[0020] A particular embodiment of the present invention includesvariants of the inventive cyanophycin synthetase, whose substratespecificity, for example, was altered, for example with regard to theproduction of polyaspartic acid, by the amino acid exchange, comparedwith the particular starting protein. The same is true for the stabilityof the enzymes of the invention in cells; for example, the enzymes haveincreased or reduced sensitivity towards degradation by proteases.

[0021] The present invention further relates to polypeptides withcyanophycin synthetase function, whose amino acid sequence has beenaltered such that they are insensitive to regulatory compounds, forexample to the metabolic endproducts regulating their activity (feedbackinsensitive).

[0022] An isolated nucleotide sequence or an isolated nucleic acidfragment means, according to the invention, an RNA or DNA polymer whichmay be single- or double-stranded and may optionally contain natural,chemically synthesized, modified or artificial nucleotides. The term“DNA polymer” here also includes genomic DNA, cDNA or mixtures thereof.

[0023] According to the invention, alleles mean functionally equivalentnucleotide sequences, i.e. nucleotide sequences with essentiallyidentical action. Functionally equivalent sequences are those sequenceswhich, despite deviating nucleotide sequences, for example due to thedegeneracy of the genetic code, still retain the desired functions.Functional equivalents thus include naturally occurring variants of thesequences described herein and also to artificial nucleotide sequencesobtained, for example, by chemical synthesis and, where appropriate,adjusted to the codon usage of the host organism. Moreover, functionallyequivalent sequences include those having a modified nucleotide sequencewhich confers on the enzyme insensitivity or resistance to inhibitors,for example.

[0024] A functional equivalent means in particular also natural orartificial mutations of an originally isolated sequence which continueto show the desired function. Mutations include substitutions,additions, deletions, exchanges or insertions of one or more nucleotideresidues. Also included here are “sense mutations” which can lead at theprotein level to the exchange of conserved amino acids, for example, butnot to any fundamental change in the protein activity and thus arefunctionally neutral. This also includes modifications of the nucleotidesequence which, at the protein level, concern the N or C terminus of aprotein but with no substantial restriction of protein function. Thesemodifications may even have a stabilizing influence on the proteinstructure.

[0025] The present invention further also includes those nucleotidesequences which are obtained by modification of the nucleotide sequence,resulting in corresponding derivatives. The aim of such a modificationmay be, for example, the further narrowing down of the coding sequencecontained therein or else, for example, the introduction of furtherrecognition sites for restriction enzymes. Functional equivalents arealso those variants whose function, compared with the starting gene orgene fragment, is reduced or enhanced.

[0026] In addition, the present invention relates to artificial DNAsequences, as long as they provide the desired properties, as describedabove, and can be inserted into or appended to the gene of thecyanophycin synthetase of the invention. It is possible, for example, todetermine such artificial DNA sequences by translating back fromproteins generated by means of computer-assisted programs (molecularmodelling) or by in vitro selection. Coding DNA sequences which havebeen obtained by translation back from a polypeptide sequence accordingto the codon usage specific for the host organism are particularlysuitable. It is possible for a skilled worker familiar with moleculargenetic methods readily to determine the specific codon usage bycomputer analyses of other, already known genes of the organism to betransformed.

[0027] According to the invention, homologous sequences mean those whichare complementary to the nucleotide sequences of the invention and/orhybridize with these sequences. The term “hybridizing sequences”includes, according to the invention, substantially similar nucleotidesequences from the group comprising DNA or RNA, which specificallyinteract with (bind to) the abovementioned nucleotide sequences underknown stringent conditions. This also includes short nucleotidesequences of, for example, from 10 to 30, preferably from 12 to 15nucleotides in length. According to the invention, “nucleotide primers”or probes are inter alia also included here.

[0028] According to the invention, the sequence regions preceding (5′ orupstream) and/or following (3′ or downstream) the coding regions(structural genes) are also included. In particular, sequence regionswith regulatory function are included here. They can influencetranscription, RNA stability or RNA processing and also translation.Examples of regulatory sequences are inter alia promoters, enhancers,operators, terminators or translation enhancers.

[0029] Operative linkage means the sequential arrangement of, forexample, promoter, coding sequence, terminator and, where appropriate,further regulatory elements, such that each of the regulatory elementscan fulfil its predetermined function when the coding sequence isexpressed. These regulatory nucleotide sequences may be of naturalorigin or can be obtained by chemical synthesis. A suitable promoter isin principle any promoter which is able to control gene expression inthe appropriate host organism. According to the invention, the saidpromoter may also be a chemically inducible promoter which makes itpossible to control at a particular time expression of the genes subjectto it in the host cell. By way of example, mention may be made here of apromoter inducible by IPTG (isopropyl β-thiogalactopyranoside).

[0030] A gene construct is prepared by fusion of a suitable promoterwith the nucleotide sequence of the invention, according to commonrecombination and cloning techniques known from the literature. The DNAfragments can be linked to one another by attaching adapters or linkersto the fragments.

[0031] Moreover, the present invention relates to a vector comprising atleast one nucleotide sequence of the type described above coding for acyanophycin synthetase specific for producing cyanophycin, regulatorynucleotide sequences operatively linked to the said nucleotide sequenceand also additional nucleotide sequences for selection of transformedhost cells, for replication within the host cell or for integration intothe appropriate host cell genome. The vector of the invention mayfurther comprise a gene construct of the abovementioned type.

[0032] Suitable vectors are those which are replicated inmicro-organisms such as, for example, bacteria, fungi and/or plants.Examples of known vectors are pBluescript (Stratagene, 11099 NorthTorney Pines Rd., La Jolla, Calif. 92 037, USA) or pET (Novagen, 601Science Drive, Madison, WJ 53 711, USA). This list, however, isnon-limiting for the present invention.

[0033] Utilizing the nucleic acid sequences of the invention, it ispossible to synthesize and use appropriate probes or else nucleotideprimers for the purpose of amplifying and isolating analogous genes fromother unicellular or multicellular organisms, preferably bacteria,fungi, algae or plants, for example with the aid of the polymerase chainreaction (“PCR”) technique.

[0034] The present invention thus also relates to a probe foridentifying and/or isolating genes coding for proteins involved incyanophycin biosynthesis, preferably further thermostable cyanophycinsynthetases; this probe is prepared starting from the inventive nucleicacid sequences of the type described above and contains a label suitablefor detection. The probe may be a section of the sequence of theinvention, for example from a conserved region, which is, for example,from 10 to 30 or, preferably, 12 to 15 nucleotides in length and whichcan hybridize specifically with homologous nucleotide sequences understringent conditions. Suitable labels are known from the literature inlarge numbers.

[0035] The present invention further relates to the transfer of theinventive nucleic acid sequence or a part thereof, coding for acyanophycin synthetase, an allele, homologue or derivative thereof, orof a nucleotide sequence hybridizing with these sequences into aheterologous host system. This also includes the transfer of a geneconstruct or vector of the invention into a heterologous host system.

[0036] According to the invention, a heterologous host system means aunicellular or multicellular organism. Examples of these aremicro-organisms, fungi, lower or higher plants, tissue or cells thereof.According to the invention, preference is given to bacteria,particularly preferably of the genus of enterobacteria and, inparticular, of the species Escherichia coli. Furthermore, useful plantssuch as potatoes or tobacco are particularly preferred.

[0037] The inventive nucleotide sequence coding for an inventivethermostable cyanophycin synthetase is transferred into one of theabovementioned host systems according to known methods. Examples ofmethods for DNA transfer into suitable host systems, which may bementioned, are transformation, electroporation, conjugation andagrobacteria-mediated DNA transfer or particle bombardment. This listserves only the purpose of illustrating the present invention and isnon-limiting.

[0038] A transformed unicellular or multicellular organism resultingfrom a successful nucleic acid transfer thus differs from thecorresponding untransformed organism by containing and being able toexpress additional nucleic acids of the inventive type.

[0039] The invention thus also relates to a transformed unicellular ormulticellular organism comprising a cyanophycin synthetase of theinvention and/or a vector comprising a cyanophycin synthetase of thetype described above.

[0040] The present invention further relates to a method for providingan inventive cyanophycin synthetase of the type described above, inwhich method the nucleotide sequence coding for the enzyme is isolatedfrom a thermophilic unicellular or multicellular organism, is, whereappropriate, operatively linked to regulatory structures and/or clonedinto a vector suitable for heterologous expression, is, whereappropriate, transferred into a heterologous host system, is expressedthere and is finally isolated from this host system and, whereappropriate, purified and/or concentrated.

[0041] Direct isolation of an amount of cyanophycin synthetase which issufficient for cyanophycin synthesis, from a thermophilic organism,without prior concentration in a heterologous system, is alsoconceivable. Furthermore, it is then possible to use the inventivecyanophycin synthetase enzyme, for example in an in vitro system forsynthesizing cyanophycin and/or secondary products thereof.

[0042] The present invention also relates to a method for producingcyanophycin and/or secondary products thereof, in which a cyanophycinsynthetase and/or a vector and/or a transformed unicellular ormulticellular organism of the type described above are used. However,the present invention includes not only the production of cyanophycinand/or secondary products thereof in a living host system but also thein-vitro synthesis of cyanophycin with the aid of an isolatedcyanophycin synthetase of the type described above.

[0043] The inventive method for producing cyanophycin is distinguishedby carrying out the enzyme-catalyzed synthesis in a temperature rangefrom 35° C. to 55° C., preferably in a range from 35° C. to 50° C.

[0044] The method of the invention is advantageously distinguished bythe fact that, owing to the wide temperature range, the process is lesserror-prone, in particular above 28° C., allows greater variability inprocess control and thus provides improved product yield. Thus, theinventive production of cyanophycin and/or secondary products thereof issubstantially more reproducible and economical than the hitherto knownmethods.

[0045] At the molecular level, the cyanophycin synthetase of theinvention catalyses an ATP-dependent chain elongation. Surprisingly, theenzyme has two active (catalytic) centres. The cyanophycin synthetase ofthe invention stepwise and alternately (sequentially) incorporates oneaspartic acid molecule and subsequently one arginine molecule into acyanophycin precursor (peptide primer). Without a primer, theenzyme-catalysed chain elongation cannot be started. Studies thereon aredepicted in FIG. 2.

[0046] Referring now to FIG. 2 there is illustrated a representation ofthe results of an SDS polyacrylamide gel electrophoresis (SDS-PAGE) forin vitro synthesis of cyanophycin-like material by means of purifiedcyanophycin synthetase. The reaction mixture contains inter alia about10 μM Primer (β-Asp-Arg)₃. After incubation for 24 hours at roomtemperature, aliquots of the reaction mixture are analysed by means ofSDS-PAGE and proteins are visualized according to standard methods. Thelanes illustrate the following: Lane 1: complete reaction mixture; lane2: reaction mixture without aspartic acid; lane 3: reaction mixturewithout arginine; lane 4: reaction mixture without ATP; lane 5: reactionmixture without primer (β-Asp-Arg)₃; lane 6: reaction mixture withheat-inactivated enzyme (5 min, 100° C.). The protein band above the97.4 kDa standard represents cyanophycin synthetase. The diffuse bandsbelow 29 kDa in lanes 1, 2 and 3 represent cyanophycin-like material.

[0047] The chemical structure of various primers used in the synthesisof cyanophycin is depicted in FIG. 1. This clearly indicates thatincorporation takes place exclusively at the C-terminal end of theprecursor and only if both amino acids, i.e. aspartic acid and arginineor another basic amino acid, are present together. A summary of thesestudies is depicted in FIG. 3 and Table 1.

[0048] Referring now to FIG. 3 there is illustrated a representation ofthe results of an SDS-PAGE for chain elongation of a primer by means ofcyanophycin synthetase at the C-terminal end of the peptide primer.Various primers (FIG. 1) are added to the reaction mixture. Afterincubation of the reaction mixtures for 24 hours at room temperature,aliquots of the reaction mixture are analysed by means of SDS-PAGE andproteins are visualized according to standard methods. The lanesillustrate the following: lane 1: mixture without primer; lane 2:mixture with about 8 μM unprotected primer (β-Asp-Arg)₃; lane 3: mixturewith about 8 μM N-terminally protected primer (ε-Ahx₂-(β-Asp-Arg)₃);lane 4: mixture with about 8 μM C-terminally protected primer((β-Asp-Arg)₃-ε-Ahx₂); lane 5: as lane 4 but with about 160 μM primer.The diffuse bands below 29 kDa in lanes 1, 2 and 3 representcyanophycin-like material. Table 1 below illustrates the cyanophycinsynthetase-catalysed incorporation of L-aspartic acid (Asp) andL-arginine (Arg) into synthetic peptide primers. TABLE 1 No. PrimerSubstrate(s) Product C-terminally blocked primer: (1)(β-Asp-Arg)₃-ε-Ahx₂ Asp + Arg none (2) (β-Asp-Arg)₃-ε-Ahx₂ Asp or Argnone N-terminally blocked primer: (3) ε-Ahx₂-(β-Asp-Arg)₃ Asp + ArgCyanophycin (4) ε-Ahx₂-(β-Asp-Arg)₃ Asp ε-Ahx₂-(β-Asp-Arg)₃-Asp (5)ε-Ahx₂-(β-Asp-Arg)₃ Arg none Unblocked primers: (6) (β-Asp-Arg)₃ Asp +Arg Cyanophycin (7) (β-Asp-Arg)₃ Asp (β-Asp-Arg)₃-Asp^(a)) (8)(β-Asp-Arg)₃ Arg none (9) (β-Asp-Arg)₃ β-Asp-Arg none (10)(β-Asp-Arg)₃-Asp Asp + Arg Cyanophycin (11) (β-Asp-Arg)₃-Asp Asp none(12) (β-Asp-Arg)₃-Asp Arg (β-Asp-Arg)₄

[0049] The present invention further relates to the use of a vectorcomprising an inventive cyanophycin synthetase of the abovementionedtype for preparing a transformed unicellular or multicellular organismas described above. The present invention likewise includes the use ofsuch a transformed unicellular or multicellular organism for producingan inventive cyanophycin synthetase and/or for producing cyanophycinand/or secondary products thereof. Moreover it is also possible to makeuse of a cyanophycin synthetase isolated according to the invention forin-vitro production of cyanophycin and/or secondary products thereof. Inaddition, the present invention relates to the use of cyanophycin and/orsecondary products thereof for producing food supplements and/orcompositions in the fields of agriculture and/or crop protection.Further fields of application for cyanophycin and/or secondary productsthereof can be found in the paper, textile, pigment, paint, ceramics,building material or detergent industry and also in the fields of waterand wastewater treatment.

[0050] The present invention is characterized in more detail by thefollowing examples which are, however, not limiting for the invention:

[0051] General Genetic Methods:

[0052] DNA isolation, plaque hybridization, polymerase chain reaction(PCR), construction of a genomic DNA gene library and the procedures forprotein analysis by means of SDS polyacrylamide gel electrophoresis(SDS-PAGE) including protein purification, as well as culturing ofmicroorganisms such as, for example, Escherichia coli are carried outaccording to standard methods described in Sambrook, J. et al. (1998,Molecular Cloning: A Laboratory Manual; 2^(nd) Edition, Cold SpringHarbor Laboratory Press, N.Y.) or according to information by themanufacturers. Blue-green algae such as, for example, Synechococcuselongatus were cultured according to descriptions in Yamaoka, T., et al.(1978, Plant Cell Physiol., 19: 943-954).

[0053] Peptide Primer Synthesis:

[0054] The branched peptide primers (β-Asp-Arg)₃, (β-Asp-Arg)₃-Asp,ε-Ahx₂-(b-Asp-Arg)₃ and (β-Asp-Arg)₃-ε-Ahx₂ (see FIG. 1;Ahx=ε-aminohexane acid) were synthesized on a solid phase followingFmoc/tBu chemistry viaO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborateactivation using the building block Fmoc-Asp-[Arg(Pmc)-OtBu]-OH. Thebuilding block was prepared in solution by the following reactionsequence: (i) acylation of H-Arg(Pmc)-OtBu (Bachem Biochemicals) withFmoc-Asp-All (All=allyl ester) (Novabiochem) usingdicyclohexylcarbodiimide/N-hydroxybenzotriazole (Novabiochem) asactivators and then (ii) opening of the allyl ester with the aid ofN-methylaniline and tetrakis(triphenylphosphine)palladium(0) ascatalyst. The synthesis is started on a resin loaded withFmoc-Arg(Pmc)-TentaGel-S-PHB (Rapp Polymere). The peptide primers arelinked by the following reaction: (i) coupling of Fmoc-Asp-OtBu andsubsequently (ii) attaching twice the building blockFmoc-Asp-[Arg-(Pmc)-OtBu]-OH. Furthermore, the N-terminally blockedpeptide primer ε-Ahx₂-(b-Asp-Arg)₃ was prepared by attachingFmoc-ε-aminohexane acid (Novabiochem) twice to the resin-bound peptideprimer described above. The finished peptides were deprotected bytreatment with 94% trifluoroacetic acid, 1% phenol, 2% water, 3%triisobutylsilane and removed from the resin, the peptides and theN-terminally blocked peptide primers being obtained as free acids. TheC-terminally blocked peptide primer (β-Asp-Arg)₃-ε-Ahx₂ was prepared ona TentaGel-SRAM resin (Rapp-Polymere) according to the followingprocedure: (i) two times coupling of Fmoc-ε-aminohexane acid andsubsequently three times coupling of the building blockFmoc-L-Asp-[L-Arg(Pmc)-OtBu). The finished primer was removed asdescribed above and gave the peptide as carboxamide.

[0055] The peptide primer (β-Asp-Arg)₃-Asp was synthesized on aTentaGel-S-PHB resin (Rapp Polymere) loaded with Fmoc-Asp(OtBu) byattaching the appropriate building block three times. As described abovethe peptide was likewise removed from the resin and deprotected. Thepeptide was obtained as free acid here. All peptide primers werepurified on a C-18 column (Vydac 201SP54) and analysed with the aid ofRP HPLC and MALDI MS.

[0056] The dipeptide β-Asp-Arg was likewise prepared on a TentaGel-S-PHBphase which had been loaded with Fmoc-Arg(Pmc) before. After the Fmocprotection group had been removed with 20% strength piperidine-DMFsolution, the resin was treated with 4 eq (equivalents) of Boc-Asp-OtBu(Bachem Chemicals), 4 eq ofO-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate and8 eq of diisopropyl-ethylamine in DMF. The peptide was removed from theresin with trifluoroacetic acid containing 1% phenol, 2% water and 3%triisobutylsilane, then precipitated with cold t-butylmethyl and finallypurified on a C-18 column (Vydac 201SP54) and analysed by RP HPLC andMALDI MS.

[0057] Reaction Mixtures and Product Analysis:

[0058] The reaction mixtures for product analysis by means of massspectrometry contain in a volume of 125 μl the following components: 100mM NH₄HCO₃ (pH 8.0), 4 mM ATP disodium salt, 20 mM MgCl₂, 8 mM KCl, 2 mMDTT, 0.2 mM L-aspartic acid, 0.2 mM L-arginine, ≧10 μM synthetic primersand 3 μg of cyanophycin synthetase.

[0059] For product analysis by means of SDS-PAGE, 125 μl of reactionmixture contain the following: 50 mM Tris-HCl (pH 8.0), 4 mM ATPdisodium salt, 20 mM MgCl₂, 20 mM KCl, 1 mM DTT, 0.8 mM L-aspartic acid,0.4 mM L-arginine, ≧10 μM synthetic primers and 3 μg of cyanophycinsynthetase.

[0060] The samples are incubated at room temperature for 10-14 hours andsubsequently either mixed with sample buffer (SDS-PAGE) or frozen (massspectrometry). The products are analysed by means of mass spectrometry(MALDI MS) according to the information in the user manual of themanufacturer (PerSeptive Biosystems).

[0061] Other than in the operating examples, or where otherwiseindicated, all numbers expressing quantities of ingredients, reactionconditions, etc. used in the specification and claims are to be understood as modified in all instance by the term “about.”

[0062] Although the invention has been described in detail in theforegoing for the purpose of illustration, it is to be understood thatsuch detail is solely for that purpose and that variations can be madetherein by those skilled in the art without departing from the spiritand scope of the invention except as it may be limited by the claims.

1 2 1 896 PRT Synechococcus elongatus 1 Met Lys Ile Leu Lys Leu Gln ThrLeu Arg Gly Pro Asn Tyr Trp Ser 1 5 10 15 Ile Arg Arg His Lys Leu IleVal Met Arg Leu Asp Leu Glu Glu Val 20 25 30 Ala Asn Thr Pro Ser Asn GlnIle Ser Gly Phe Val Asp Gly Leu Val 35 40 45 Arg Val Leu Pro Ser Leu TyrAsn His Phe Cys Ser Leu Gly His Glu 50 55 60 Gly Gly Phe Leu Thr Arg LeuArg Glu Gly Thr Tyr Leu Gly His Val 65 70 75 80 Val Glu His Val Ala LeuGlu Leu Gln Glu Leu Ala Gly Met Pro Val 85 90 95 Gly Phe Gly Arg Thr ArgGlu Thr Ser Thr Pro Gly Val Tyr Gln Val 100 105 110 Val Tyr Glu Tyr GlnVal Glu Glu Ala Gly Arg Tyr Ala Gly Arg Ala 115 120 125 Ala Val Arg LeuCys Gln Ser Ile Ile Asp Thr Gly Thr Tyr Pro Gln 130 135 140 Gln Glu LeuAsp Gln Asp Leu Ala Asp Leu Arg Glu Leu Lys Ala Lys 145 150 155 160 AlaSer Leu Gly Pro Ser Thr Glu Ala Ile Val Arg Glu Ala Glu Ala 165 170 175Arg Asn Ile Pro Trp Phe Glu Leu Ser Ser Arg Ser Ile Ile Gln Leu 180 185190 Gly Tyr Gly Ala Arg Ser His Arg Met Gln Ala Thr Leu Ser Asp Arg 195200 205 Ser Ser Ile Leu Ala Val Glu Leu Ala Ser Asp Lys Glu Gly Ala Lys210 215 220 Arg Leu Leu Gln Asp Ala Gly Ile Pro Val Pro Lys Gly Thr ValIle 225 230 235 240 Arg Tyr Ile Glu Asp Leu Pro Glu Ala Ile Glu Glu IleGly Gly Tyr 245 250 255 Pro Ile Val Ile Lys Pro Leu Asn Gly Asn His GlyArg Gly Ile Thr 260 265 270 Ile Asp Ile Asn Ser Leu Glu Ala Ala Glu GluAla Phe Glu Ile Ala 275 280 285 Ser Ser Ile Ser Lys Ser Val Ile Val GluArg Tyr His Ala Gly Arg 290 295 300 Asp Phe Arg Val Leu Val Val Asn GlyLys Val Val Ala Val Ala Glu 305 310 315 320 Arg Val Pro Ala His Val IleGly Asp Gly His Ser Thr Ile Glu Glu 325 330 335 Leu Ile Glu Lys Thr AsnGln Asp Pro Gln Arg Gly Asp Gly His Asp 340 345 350 Asn Ile Leu Thr ArgIle Glu Val Asn His Asp Thr Trp Thr Leu Leu 355 360 365 Glu Lys Gln GlyTyr Thr Leu Asn Thr Val Leu Gln Pro Gly Glu Ile 370 375 380 Cys Tyr LeuArg Ala Thr Ala Asn Leu Ser Thr Gly Gly Ile Ala Ile 385 390 395 400 AspArg Thr Asp Glu Ile His Pro Glu Asn Val Trp Ile Cys Gln Arg 405 410 415Ala Ala Arg Ile Ile Gly Leu Asp Ile Ala Gly Ile Asp Val Val Ser 420 425430 Pro Asp Ile Ser Gln Pro Leu Ser Lys Val Gly Gly Val Ile Val Glu 435440 445 Val Asn Ala Ala Pro Gly Phe Arg Met His Thr Asn Pro Ser Gln Gly450 455 460 Ile Ala Arg Asn Val Ala Glu Pro Val Leu Asn Met Leu Phe ProPro 465 470 475 480 Gly Thr Pro Cys Arg Ile Pro Ile Phe Ala Ile Thr GlyThr Asn Gly 485 490 495 Lys Thr Thr Thr Thr Arg Leu Ile Ala His Ile CysLys Gln Thr Gly 500 505 510 Gln Thr Val Gly Tyr Thr Thr Thr Asp Gly IleTyr Ile Gly Asp Tyr 515 520 525 Leu Val Glu Lys Gly Asp Thr Thr Gly ProGln Ser Ala Gln Leu Ile 530 535 540 Leu Gln Asp Pro Thr Val Glu Ile AlaVal Leu Glu Thr Ala Arg Gly 545 550 555 560 Gly Ile Leu Arg Ser Gly LeuGly Phe Asp His Cys Asp Val Gly Val 565 570 575 Val Leu Asn Val Gln AlaAsp His Leu Gly Leu Gly Asp Ile Asp Thr 580 585 590 Val Glu Gln Leu AlaAsp Leu Lys Ala Val Val Val Glu Ser Ala Trp 595 600 605 Pro Asn Gly TyrAla Val Leu Asn Ala Asp Asp Pro Leu Val Ala Ala 610 615 620 Met Ala ArgGln Val Lys Ala Gln Val Ala Tyr Phe Ser Met Asp Pro 625 630 635 640 HisAsn Pro Ile Ile Arg Gln His Ile Gln Gln Gly Gly Leu Ala Ala 645 650 655Val Tyr Glu Asn Gly Tyr Leu Ser Ile Leu Lys Gly Asp Trp Thr Leu 660 665670 Arg Ile Glu Gln Ala Glu Asn Val Pro Ile Thr Leu Gly Ala Arg Ala 675680 685 Ser Phe Met Ile Ala Asn Ala Leu Ala Ala Ser Leu Ala Ala Phe Ala690 695 700 Gln Gly Ile Ser Ile Glu His Ile Arg Ala Ala Leu Thr Thr PheArg 705 710 715 720 Thr Ser Val Glu Gln Thr Pro Gly Arg Met Asn Leu PheAsp Leu Gly 725 730 735 Gln Phe Ser Val Leu Val Asp Tyr Ala His Asn ProAla Gly Tyr Glu 740 745 750 Ala Ile Gly Glu Phe Val Gln Lys Trp Pro GlyGln Arg Ile Gly Val 755 760 765 Val Gly Gly Pro Gly Asp Arg Arg Asp GlnAsp Leu Glu Gln Leu Gly 770 775 780 Glu Leu Ser Ala Lys Ile Phe Asp TrpIle Ile Ile Lys Glu Asp Asp 785 790 795 800 Asp Thr Arg Gly Arg Pro ArgGly Asp Ala Ala Tyr Trp Ile Glu Arg 805 810 815 Gly Val His His His SerVal Gln Arg Gln Tyr Asp Ile Ile His Asp 820 825 830 Glu Val Ala Ala IleGln Phe Ala Leu Asp Arg Ala Pro Lys Gly Ser 835 840 845 Leu Val Val IlePhe Pro Ala Glu Val Ser Arg Thr Ile Gln Leu Ile 850 855 860 Arg Gln HisHis Gln Arg Leu Gln Gly Glu Thr Ile Asn Gly Phe His 865 870 875 880 SerGlu Gly Arg Pro Thr Ser Gly Asp Leu Asn Pro Ser Ile Phe His 885 890 8952 2691 DNA Synechococcus elongatus CDS (1)..(2691) cphA (cyanophycinsynthetase) 2 atg aag att ctc aaa tta caa acg ctg cgg ggt ccc aat tactgg agc 48 Met Lys Ile Leu Lys Leu Gln Thr Leu Arg Gly Pro Asn Tyr TrpSer 1 5 10 15 att cgg cgt cat aag ctg att gtc atg cgt tta gat cta gaagag gtg 96 Ile Arg Arg His Lys Leu Ile Val Met Arg Leu Asp Leu Glu GluVal 20 25 30 gcc aac acc ccc tcc aat cag att tct ggg ttt gtg gat ggg ttggtg 144 Ala Asn Thr Pro Ser Asn Gln Ile Ser Gly Phe Val Asp Gly Leu Val35 40 45 cgg gtt ttg ccg agt ctt tac aat cat ttt tgt tct ctc gga cac gaa192 Arg Val Leu Pro Ser Leu Tyr Asn His Phe Cys Ser Leu Gly His Glu 5055 60 ggg ggc ttt ctc acc cgc ctc cga gaa ggt acg tat ctt ggt cat gtg240 Gly Gly Phe Leu Thr Arg Leu Arg Glu Gly Thr Tyr Leu Gly His Val 6570 75 80 gtt gaa cat gtt gcc ctc gag ctc caa gaa ctg gca ggg atg ccc gtt288 Val Glu His Val Ala Leu Glu Leu Gln Glu Leu Ala Gly Met Pro Val 8590 95 ggc ttt ggc cgc acg cgg gag acc tca acg ccg ggg gtg tat caa gtg336 Gly Phe Gly Arg Thr Arg Glu Thr Ser Thr Pro Gly Val Tyr Gln Val 100105 110 gtc tat gaa tac caa gtg gaa gaa gcg ggc cgc tat gcc ggc cga gca384 Val Tyr Glu Tyr Gln Val Glu Glu Ala Gly Arg Tyr Ala Gly Arg Ala 115120 125 gca gtg cga ctg tgc caa agt att att gat acg ggt acc tat ccc cag432 Ala Val Arg Leu Cys Gln Ser Ile Ile Asp Thr Gly Thr Tyr Pro Gln 130135 140 caa gaa ctg gat cag gat ctc gcc gat ctc cgg gag ttg aaa gca aaa480 Gln Glu Leu Asp Gln Asp Leu Ala Asp Leu Arg Glu Leu Lys Ala Lys 145150 155 160 gcc tcc ctt ggc ccg agt acg gaa gcg att gtc cgc gaa gcc gaagcc 528 Ala Ser Leu Gly Pro Ser Thr Glu Ala Ile Val Arg Glu Ala Glu Ala165 170 175 cgc aac atc cct tgg ttt gag ttg agc agt cgc tcg att att caattg 576 Arg Asn Ile Pro Trp Phe Glu Leu Ser Ser Arg Ser Ile Ile Gln Leu180 185 190 ggc tat ggc gcc cgc agt cat cgg atg caa gcc aca ttg agc gatcgc 624 Gly Tyr Gly Ala Arg Ser His Arg Met Gln Ala Thr Leu Ser Asp Arg195 200 205 agt agc atc ttg gca gtt gaa ctc gcc agt gac aaa gaa ggg gcaaag 672 Ser Ser Ile Leu Ala Val Glu Leu Ala Ser Asp Lys Glu Gly Ala Lys210 215 220 cga ctg ctt cag gat gcg gga att ccc gtg cct aag gga acc gtcatc 720 Arg Leu Leu Gln Asp Ala Gly Ile Pro Val Pro Lys Gly Thr Val Ile225 230 235 240 cgc tat att gaa gac ctc ccc gag gcc att gag gag atc ggtggc tat 768 Arg Tyr Ile Glu Asp Leu Pro Glu Ala Ile Glu Glu Ile Gly GlyTyr 245 250 255 ccc att gtc att aag ccc ctc aac ggc aac cac ggt cgc gggatt acg 816 Pro Ile Val Ile Lys Pro Leu Asn Gly Asn His Gly Arg Gly IleThr 260 265 270 att gac atc aac agc cta gaa gca gcc gaa gaa gcc ttt gaaatt gcc 864 Ile Asp Ile Asn Ser Leu Glu Ala Ala Glu Glu Ala Phe Glu IleAla 275 280 285 agc agc atc tcc aaa tcc gtc att gtg gaa cgc tat cat gccggt cgc 912 Ser Ser Ile Ser Lys Ser Val Ile Val Glu Arg Tyr His Ala GlyArg 290 295 300 gac ttc cgc gtt cta gtg gtc aat ggc aaa gtg gtt gct gttgct gaa 960 Asp Phe Arg Val Leu Val Val Asn Gly Lys Val Val Ala Val AlaGlu 305 310 315 320 cgg gtg ccg gcc cat gtg att ggc gat ggc cac tcc accatc gaa gaa 1008 Arg Val Pro Ala His Val Ile Gly Asp Gly His Ser Thr IleGlu Glu 325 330 335 ctc att gag aaa acg aac caa gac ccg caa cgg gga gacggt cac gat 1056 Leu Ile Glu Lys Thr Asn Gln Asp Pro Gln Arg Gly Asp GlyHis Asp 340 345 350 aat atc ctc acc cgc att gaa gtc aac cac gac act tggaca ctc ctg 1104 Asn Ile Leu Thr Arg Ile Glu Val Asn His Asp Thr Trp ThrLeu Leu 355 360 365 gaa aaa cag ggc tat acc ctg aat acg gtc ttg caa ccgggg gaa att 1152 Glu Lys Gln Gly Tyr Thr Leu Asn Thr Val Leu Gln Pro GlyGlu Ile 370 375 380 tgt tat cta cgg gcc acg gcg aac cta agt act ggt ggcatt gcc atc 1200 Cys Tyr Leu Arg Ala Thr Ala Asn Leu Ser Thr Gly Gly IleAla Ile 385 390 395 400 gat cgc act gat gaa att cac ccg gaa aat gtt tggatt tgc cag cgg 1248 Asp Arg Thr Asp Glu Ile His Pro Glu Asn Val Trp IleCys Gln Arg 405 410 415 gct gct cgg atc att ggc ctc gat att gct ggt attgac gtt gtc agc 1296 Ala Ala Arg Ile Ile Gly Leu Asp Ile Ala Gly Ile AspVal Val Ser 420 425 430 ccc gat att agt cag ccc ctg tct aaa gtt ggc ggtgtg att gtc gag 1344 Pro Asp Ile Ser Gln Pro Leu Ser Lys Val Gly Gly ValIle Val Glu 435 440 445 gtc aat gcc gct cct ggc ttt cgc atg cac acc aacccc agc caa ggg 1392 Val Asn Ala Ala Pro Gly Phe Arg Met His Thr Asn ProSer Gln Gly 450 455 460 att gcc cgc aat gtt gcc gaa ccg gtg ttg aat atgctc ttt cca ccg 1440 Ile Ala Arg Asn Val Ala Glu Pro Val Leu Asn Met LeuPhe Pro Pro 465 470 475 480 gga aca cct tgc cgc atc ccg atc ttt gcc attacg ggg acc aat ggc 1488 Gly Thr Pro Cys Arg Ile Pro Ile Phe Ala Ile ThrGly Thr Asn Gly 485 490 495 aaa acc acc acc acc cgt ctc att gcc cat atctgc aaa caa acg ggg 1536 Lys Thr Thr Thr Thr Arg Leu Ile Ala His Ile CysLys Gln Thr Gly 500 505 510 caa acc gtt ggc tac acc acc aca gac ggc atctat att ggc gat tat 1584 Gln Thr Val Gly Tyr Thr Thr Thr Asp Gly Ile TyrIle Gly Asp Tyr 515 520 525 ctg gtg gaa aaa gga gac acc acc ggc ccc caaagt gcc caa ctg atc 1632 Leu Val Glu Lys Gly Asp Thr Thr Gly Pro Gln SerAla Gln Leu Ile 530 535 540 ctg cag gac ccc acc gtt gag atc gcc gtt ctcgaa acg gcg cga ggt 1680 Leu Gln Asp Pro Thr Val Glu Ile Ala Val Leu GluThr Ala Arg Gly 545 550 555 560 ggg att ctc cgc tcc ggc ttg ggc ttt gaccat tgt gat gtc ggg gtg 1728 Gly Ile Leu Arg Ser Gly Leu Gly Phe Asp HisCys Asp Val Gly Val 565 570 575 gtg ctc aat gtg cag gct gat cac ctt ggcctt ggc gat att gac acc 1776 Val Leu Asn Val Gln Ala Asp His Leu Gly LeuGly Asp Ile Asp Thr 580 585 590 gtt gag cag ttg gcg gac tta aag gca gtggtg gtg gaa tct gct tgg 1824 Val Glu Gln Leu Ala Asp Leu Lys Ala Val ValVal Glu Ser Ala Trp 595 600 605 cca aat ggc tac gct gtg ttg aat gcc gatgat ccc cta gtg gcg gca 1872 Pro Asn Gly Tyr Ala Val Leu Asn Ala Asp AspPro Leu Val Ala Ala 610 615 620 atg gca cgc caa gtc aaa gct caa gtg gcctat ttc tcg atg gat ccc 1920 Met Ala Arg Gln Val Lys Ala Gln Val Ala TyrPhe Ser Met Asp Pro 625 630 635 640 cac aat ccc atc att cgg cag cac atccag cag ggg gga ctc gcc gct 1968 His Asn Pro Ile Ile Arg Gln His Ile GlnGln Gly Gly Leu Ala Ala 645 650 655 gtt tat gaa aat ggc tac ctc tca attttg aaa ggg gac tgg aca ctg 2016 Val Tyr Glu Asn Gly Tyr Leu Ser Ile LeuLys Gly Asp Trp Thr Leu 660 665 670 cgc att gag cag gca gaa aat gtg cccatt acc ctt ggc gct cga gca 2064 Arg Ile Glu Gln Ala Glu Asn Val Pro IleThr Leu Gly Ala Arg Ala 675 680 685 agc ttt atg att gcc aat gcc ctc gctgcc agt cta gcg gcc ttt gcc 2112 Ser Phe Met Ile Ala Asn Ala Leu Ala AlaSer Leu Ala Ala Phe Ala 690 695 700 caa ggc atc agt att gag cat att cgcgcc gcc ttg acc acc ttc cga 2160 Gln Gly Ile Ser Ile Glu His Ile Arg AlaAla Leu Thr Thr Phe Arg 705 710 715 720 acc tcg gtg gag caa acc ccc ggtcgg atg aac ctc ttt gat ttg ggg 2208 Thr Ser Val Glu Gln Thr Pro Gly ArgMet Asn Leu Phe Asp Leu Gly 725 730 735 caa ttt agt gtc ttg gtg gac tatgcc cac aat cca gca ggg tat gaa 2256 Gln Phe Ser Val Leu Val Asp Tyr AlaHis Asn Pro Ala Gly Tyr Glu 740 745 750 gcc att ggt gaa ttt gtc cag aaatgg cca ggg cag cgc att ggt gtc 2304 Ala Ile Gly Glu Phe Val Gln Lys TrpPro Gly Gln Arg Ile Gly Val 755 760 765 gtt ggc gga cca ggc gat cgc cgcgat caa gac ttg gag caa ctg ggg 2352 Val Gly Gly Pro Gly Asp Arg Arg AspGln Asp Leu Glu Gln Leu Gly 770 775 780 gaa ctc tcg gcg aaa att ttt gattgg atc atc att aag gaa gat gat 2400 Glu Leu Ser Ala Lys Ile Phe Asp TrpIle Ile Ile Lys Glu Asp Asp 785 790 795 800 gat acc cgt ggc cgg cct cggggc gat gcc gcc tat tgg att gag cgg 2448 Asp Thr Arg Gly Arg Pro Arg GlyAsp Ala Ala Tyr Trp Ile Glu Arg 805 810 815 ggg gta cat cac cac agt gtccag cgg caa tac gac atc atc cat gac 2496 Gly Val His His His Ser Val GlnArg Gln Tyr Asp Ile Ile His Asp 820 825 830 gag gtg gca gcg att caa tttgcc ctc gat cgc gct ccc aaa gga tcc 2544 Glu Val Ala Ala Ile Gln Phe AlaLeu Asp Arg Ala Pro Lys Gly Ser 835 840 845 tta gtg gtg atc ttt cca gcggaa gtc agc cgc acg att caa ctg att 2592 Leu Val Val Ile Phe Pro Ala GluVal Ser Arg Thr Ile Gln Leu Ile 850 855 860 cgc cag cat cac caa cga ctccaa ggg gaa acg atc aat ggc ttt cac 2640 Arg Gln His His Gln Arg Leu GlnGly Glu Thr Ile Asn Gly Phe His 865 870 875 880 agt gag gga agg ccc accagt ggt gat ctc aac ccc tcc atc ttt cat 2688 Ser Glu Gly Arg Pro Thr SerGly Asp Leu Asn Pro Ser Ile Phe His 885 890 895 tag 2691

We claim:
 1. A cyanophycin synthetase comprising an amino acid sequenceaccording to SEQ ID No: 01, encoded by a nucleotide sequence accordingto SEQ ID No: 02, an allele, homologue or derivative of said nucleotidesequence or a nucleotide sequence hybridizing therewith, and originatingfrom Synechococcus elongatus, wherein said cyanophycin synthetase has atemperature optimum of at least 35° C.
 2. A vector comprising at leastone nucleotide sequence coding for a cyanophycin synthetase according toclaim 1 or 19, which is specific for cyanophycin production.
 3. Atransformed unicellular or multicellular organism comprising acyanophycin synthetase according to claim 19 and/or a vector accordingto claim
 2. 4. A transformed unicellular or multicellular organismaccording to claim 3, wherein said organism is selected from the groupconsisting of a microorganism, a fungus, a lower or higher plant, tissueor at least one cell therefrom.
 5. A method for providing a cyanophycinsynthetase according to claim 1 or 19, comprising the steps ofoperatively linking said nucleotide sequence coding for said cyanophycinsynthetase to regulatory structures and/or is cloning said nucleotidesequence into a vector suitable for heterologous expression,transferring said nucleotide sequence into a heterologous host systemand expressing and isolating and/or purifying and/or concentrating saidcyanophycin synthetase from said host system.
 6. A method for producingcyanophycin and the secondary products to be produced therefrom,comprising the step of employing a cyanophycin synthetase according toclaim 1 or 19 and/or a vector according to claim 2 and/or a transformedunicellular or multicellular organism according to any of claims 3 and 4to produce said cyanophycin.
 7. A method for preparing a transformedunicellular or multicellular organism according to either of claims 3and 4 comprising the step of employing a vector according to claim 2 toproduce said transformed organism.
 8. A method for producing a compoundselected from the group consisting of a cyanophycin synthetase accordingto claim 1 or 19 and cyanophycin comprising the steps of providing thetransformed unicellular or multicellular organism according to any ofclaims 3 to 4, whereupon said organism produces said cyanophycinsynthetase and/or said cyanophycin.
 9. A method for producingcyanophycin comprising the steps of providing said cyanophycinsynthetase according to claim 1 or 19, and employing said cyanophycinsynthetase to produce said cyanophycin.
 10. Isoenzymes and modifiedforms of cyanophycin synthetase wherein said isoenzymes and saidmodified forms of cyanophycin synthetase are obtained by modifyingcyanophycin synthetasefrom Synechococcus elongatus and wherein said isoenzymes and modified forms of cyanophycin synthetase have a temperatureoptimum in the range of 35° C. to 55° C.
 11. The isoenzymes and themodified forms of cyanophycin synthetase according to claim 10, whereinsaid isoenzymes and said modified forms of cyanophycin synthetase areobtained by amino acid exchange.
 12. The isoenzymes and the modifiedforms of cyanophycin synthetase according to claim 11, wherein the aminoacid exchange is carried out by modifying a nucleotide sequence of anunderlying gene of said Synechococcus elongatus.
 13. An artificial DNAsequence, comprising an artificial DNA sequence that is insertable intoor appendable to a gene wherein said artificial DNA sequence encodes forand expresses the cyanophycin synthetase according to claim 1 or
 19. 14.A probe for the identification and/or isolation of one or more genescoding for proteins involved in cyanophycin biosynthesis, said probecomprising a label suitable for detecting cyanophycin synthetase andmodifications thereof according to claims 1 and
 10. 15. A heterologoushost system comprising a nucleic acid sequence or a part thereof codingfor a member of the group consisting of cyanophycin synthetase, anisoenzyme and modified forms thereof according to claims 1, 19 or 10.16. A method for the synthesis of polyaspartic acid or argininecomprising the steps of providing said cyanophycin synthetase accordingto claim 1 or 19 and employing said cyanophycin synthetase to producesaid polyaspartic acid or said arginine.
 17. Cyanophycin synthetasecomprising a DNA sequence selected from the group consisting of anatural DNA sequence and an artificial DNA sequence, said DNA sequencebeing located between the 5′ or upstream and/or 3′ or downstreamposition of the cyanophycin synthetase according to claim 1, whereinsaid DNA sequences influence transcription, RNA stability of RNAprocessing, and translation.
 18. The synthetase of claim 1 wherein saidsynthetase has a temperature optimum in the range of about 35° C. toabout 55° C.
 19. The synthetase of claim 1 wherein said synthetase has atemperature optimum in the range of 35° C. to 55° C.
 20. The synthetaseof claim 1 wherein said synthetase has a temperature optimum in therange of 35° C. to 50° C.
 21. A polypeptide having cyanophycinsynthetase functionality comprising a cyanophycin synthetase having atemperature optimum of at least 35° C. and wherein at least one aminoacid sequence of said polypeptide has been altered such that saidpolypeptide is rendered insensitive to the regulating action ofregulatory compounds that would otherwise regulate the activity of saidpolypeptide with respect to said polypeptide's cyanophycin synthetasefunctionality.
 22. The polypeptide of claim 21 wherein said cyanophycinsynthetase is a cyanophycin synthetase according to claims 1 or 19.